UCSF Diabetes Center. NIH UCSF DERC Islet Core. JDRF. LifeScan Inc. Johnson Johnson &amp; Company.

Preparazione di isole per trapianto (Tx) <ol><li> Sotto un microscopio invertito, selezionare manualmente isolette con una Pipetman P200 e punta della pipetta direttamente dalla isole coltivate in un piatto di 100mm. </li><li> Conte isolotti 100 in una volta e trasferire in ogni provetta (~ 500 isolotti / tubo / mouse). </li><li> Lasciare isolotti di depositarsi sul fondo dei tubi microcentrifuga. </li><li> Disegna uno isolotto a pellet in un Pipetman P200 (fissata a 130ul) utilizzando una scala a parete sottile punta della pipetta. </li><li> Posizionare un adattatore tubo di silicone sopra la punta della siringa. Inserire una lunghezza di PE50 tubo nel adattatore silicone. </li><li> Appendere la Pipetman a lato del cofano e il nastro PE50 tubo alla parete cappa superiore alla isolotti nella punta. In questo modo il isolette di stabilirsi solo nella punta della pipetta. </li><li> Trasferire il isolotti nel tubo PE50 lentamente componendo il Pipetman e spostando gli isolotti nel tubo PE50, facendo attenzione a non espellere le isole dalla fine. </li><li> Fare un nodo alla fine smussata del tubo PE50. Pur mantenendo le piegare, staccare il tubo in silicone adattatore dal Pipetman e fissare la piega con l&#39;adattatore in silicone. </li><li> Posizionare le isole, nel tubo PE50, piegato lato tubi di silicone adattatore verso il basso, in un mL 15 conico con una pipetta 5 ml e taglio del nastro il tubo PE50 sul lato della pipetta 5 mL per evitare di curling del tubo PE50 mentre centrifugazione. </li><li> Centrifugare il conicals PE50/15mL a 1000 giri al minuto e spegnere centrifuga. (Non preparare più di 10 preparati isolotto in una sola volta). </li><li> Tubo posto sul ghiaccio. </li></ol> Preparazione del mouse per trapianti <ol><li> Anestetizzare i topi con isofluorane o con ketamina / Xylexene (vedi protocollo di preparazione). </li><li> Dopo che ha preso effetto anestetico, radere il fianco sinistro del mouse. </li><li> Tampone pelle di topo, centro-out, con tampone Iodio Povidone e poi pulire con un tampone imbevuto ETOH. </li><li> Individuare il rene sinistro (appena a destra della milza). Fai una piccola incisione nella pelle, esponendo il peritoneo. </li><li> Fai una piccola incisione nel peritoneo esporre il rene. Mantenere la piccola incisione aiuterà a mantenere il rene sollevato ed esposto. </li><li> Applicare una leggera pressione su entrambi i lati dell&#39;incisione, alzare o pop il rene fuori del mouse. </li><li> Mantenere umido il renali mediante l&#39;applicazione di soluzione fisiologica con un tampone di cotone con punta. </li><li> Utilizzando una siringa 23 o 25 gauge, fare un piccolo graffio sul fianco destro del rene, la creazione di un nick nella capsula del rene, non troppo profonda o troppo grande. </li></ol> Trapianto di isole Nota: Quando il mouse si sta preparando per Tx, la seconda persona deve preparare la vite-drive Hamilton siringa trapianto di isole. <ol><li> Rimuovere lentamente il tubo in silicone adattatore dal tubo PE50 mantenendo il nodo nel tubo. </li><li> Posizionare l&#39;estremità opposta del tubo PE50 nel tubo in silicone adattatore e posizionare il tubo in silicone adattatore sulla punta della pipetta collegata al &quot;vite-drive&quot; siringa di vetro Hamilton. Rilasciare lentamente il nodo nel tubo PE50, assicurandosi che le isole non fuoriesca. </li><li> Anticipo le isolette lentamente fino alla punta del tubo PE50 utilizzando il meccanismo &quot;a vite&quot;, ma mantenere una piccola bolla d&#39;aria nella parte anteriore delle isole nel tubo PE50. </li><li> Nel nick fatto nel rene, far scorrere con attenzione la PE50 tubi sotto la capsula, facendo una piccola tasca. State molto attenti a non sgorbia o pungere il rene attraverso la capsula. </li><li> Aiuta a mantenere la zona umida e capsula con normale soluzione fisiologica Gentamicina tampone di cotone imbevuto di punta, una capsula secca si strappa facilmente. </li><li> Spostare delicatamente il tubo in tutte le direzioni, creando una &quot;tasca&quot; per le isole trapiantate per riposare. Ricordarsi di riapplicare ulteriore soluzione fisiologica per mantenere la zona umida. </li><li> Sotto la direzione della persona che ha aperto il mouse e che ha posto il PE50 tubi sotto la capsula renale, la seconda persona, che ha preparato il mouse e la siringa trapianto Hamilton, lentamente anticipo isole sotto la capsula, all&#39;interno della &quot;tasca&quot;, dietro una piccola bolla d&#39;aria formulato dal tubo PE50, fino a quando tutte le isole sono trapiantati. </li><li> Rimuovere lentamente il tubo PE50, asciugare la zona con un tampone asciutto e con attenzione cauterizzare il nick a basse temperature. </li><li> Utilizzando un tampone di cotone asciutto capovolto, assicurarsi che tutti sanguinamento si è fermato. Una volta emorragia si è fermata, ri-inumidire il rene con soluzione salina sterile, e gentilmente sostituire il rene nel peritoneo prima di chiudere il mouse con sutura e punti metallici pelle. </li></ol> Chiusura / Revival di mouse <ol><li> Chiudere il peritoneo con un punto in esecuzione utilizzando 5-0 suture di seta w / un ago C-6 19mm. </li><li> Forcipe utilizzando disegnare entrambi i lati della incisione cutanea insieme. </li><li> Il fiocco pelle con 2 o 3 punti. </li><li> Pulire la pelle del mouse di qualsiasi sangue, usando un tampone di cotone con punta e Saline. </li><li> Immediatamente trattare il mouse con una iniezione sottocutanea di Flunixin e buprenorfina. </li><li> Posizionare il mouse in una gabbia, che viene posizionato su una piastra elettrica o sotto una lampada riscaldante, fino a quando il mouse è pienamente attiva. </li><li> Rimuovere i punti della pelle in 2 settimane. </li></ol>

<ol>
	<li>Tang, Q., Henriksen, K. J., Bi, M., Finger, E. B., Szot, G., Ye, J., Masteller, E. L., McDevitt, H., Bonyhadi, M., Bluestone, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro-expanded+antigen-specific+regulatory+T+cells+suppress+autoimmune+diabetes.">In vitro-expanded antigen-specific regulatory T cells suppress autoimmune diabetes.</a> J Exp Med. 199, 1455-1465 (2004).</li><li>Rulifson, I. C., Szot, G. L., Palmer, E., Bluestone, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inability+to+induce+tolerance+through+direct+antigen+presentation.">Inability to induce tolerance through direct antigen presentation.</a> Am J Transplant. 2, 510-519 (2002).</li><li>Lenschow, D. J., Zeng, Y., Hathcock, K. S., Zuckerman, L. A., Freeman, G., Thistlethwaite, J. R., Gray, G. S., Hodes, R. J., Bluestone, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+transplant+rejection+following+treatment+with+anti-B7-2+and+anti-B7-1+antibodies.+Transplantation.">Inhibition of transplant rejection following treatment with anti-B7-2 and anti-B7-1 antibodies. Transplantation.</a> Transplantation. 60, 1171-1178 (1995).</li><li>Lenschow, D. J., Zeng, Y., Thistlethwaite, J. R., Montag, A., Brady, W., Gibson, M. G., Linsley, P. S., Bluestone, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+survival+of+xenogeneic+pancreatic+islet+grafts+induced+by+CTLA4lg.">Long-term survival of xenogeneic pancreatic islet grafts induced by CTLA4lg.</a> Science. 257, 789-792 (1992).</li></ol>

Questo protocollo fornisce una soluzione pratica ed efficiente per fornire in modo pulito e facilmente le cellule o isolotti sotto la capsula renale di topi diabetici o normale. La tecnica di concentrazione e cubettatura le isolette o cellule nel tubo di consegna finale (PE50) utilizzato per il trapianto di cellule sotto la capsula renale offre un metodo semplice ed efficace di trapianto di cellule, riducendo qualsiasi sforzo eccessivo per le celle o per il mouse.

<table><tbody><tr><td>Surgical gloves</td><td>Fisher Scientific</td><td>11-394-95(sz)</td><td></td></tr><tr><td>Surgical scrub sponges</td><td>Moore Medical</td><td>42940AK</td><td></td></tr><tr><td>Forceps</td><td>Miltex Inc.</td><td>6-114 and 6-26</td><td>2 with teeth, 2 straight</td></tr><tr><td>Dissecting scissors n=2</td><td>Miltex Inc.</td><td>5-290</td><td></td></tr><tr><td>Oster razor (sz. 40 blade)</td><td>Fisher Scientific</td><td>01-305-10B</td><td></td></tr><tr><td>4x4 Sterile gauze pads</td><td>Moore Medical</td><td>08252AK</td><td></td></tr><tr><td>Povidone Iodine pads</td><td>Moore Medical</td><td>08486AK</td><td></td></tr><tr><td>Alcohol pads</td><td>Fisher Scientific</td><td>14-819-2</td><td></td></tr><tr><td>Heating pad</td><td>Moore Medical</td><td>42508AK</td><td></td></tr><tr><td>23G or 25G needles</td><td></td><td></td><td>1&quot; long</td></tr><tr><td>Cidex solution</td><td>Moore Medical</td><td>07535AK</td><td>for cleaning/santizing instruments</td></tr><tr><td>PE50 polyethylene tubing</td><td>BD Biosciences</td><td>427411</td><td>PE50, 0.965mm O.D. x 0.58mm I.D.</td></tr><tr><td>Cidex + 28 day soln</td><td>Moore Medical</td><td>35625AK</td><td></td></tr><tr><td>Silicone tubing</td><td>Spectrum Chromat.</td><td>123732</td><td>5/32&amp;#8221;OD x 1/32&amp;#8221; ID</td></tr><tr><td>Instrument sterilizing container</td><td>Moore Medical</td><td>39074AK</td><td></td></tr><tr><td>Straight Pipet Tips</td><td>USA Scientific, Inc.</td><td>1111-0810</td><td>sterile</td></tr><tr><td>Glass syringe w/screw-drive</td><td>Hamilton Co</td><td>1001</td><td></td></tr><tr><td>Cauterizing tool</td><td>Roboz Surgical Instruments Co.</td><td>RS230</td><td></td></tr><tr><td>Needle Holder</td><td>Moore Medical</td><td>41-067</td><td></td></tr><tr><td>5-0 silk</td><td>LOOK Surgical</td><td>754B</td><td>for suture (6-C) </td></tr><tr><td>Cotton tipped swab</td><td>Moore Medical</td><td></td><td></td></tr><tr><td>9mm autoclip stapler</td><td>BD Biosciences</td><td>7630</td><td></td></tr><tr><td>9mm staples</td><td>BD Biosciences</td><td>7631</td><td></td></tr><tr><td>9mm staple remover</td><td>BD Biosciences</td><td>7637</td><td></td></tr></tbody></table>

transplantation of pancreatic islets into the kidney capsule of diabetic mice

Il nostro protocollo è stato sviluppato per fornire in modo pulito e facilmente le cellule o isolotti sotto la capsula renale di topi diabetici o normale. Abbiamo scoperto che era più facile concentrare le isolette o cellule in pellet nel tubo di consegna finale (PE50) utilizzato per il trapianto di cellule sotto la capsula renale. Questa tecnica fornisce velocità e facilità, riducendo qualsiasi sforzo eccessivo per le celle o per il mouse. Caricamento in corso: Fondata, raccolte a mano, isolotti o cellule pellet sono accuratamente aspirato dal fondo di una provetta 1,5 ml con un pipetteman P200 e un rettilineo, a parete sottile punta della pipetta. Una lunghezza di PE50 tubo è attaccato alla punta della pipetta con un piccolo tubo di silicone adattatore. Le cellule sono permesso di stabilirsi, in punta, e poi vengono trasferiti al tubo PE50 lentamente componendo il pipetteman. Una volta che le cellule sono quasi alla fine del tubo PE50, un punto di discontinuità è fatto e il tubo in silicone adattatore è posizionato sopra il nodo. La PE50 tubo è trasferito ad un 15 ml conica contenente una riduzione del 5 pipetta ml, e il tubo PE50 è registrato sul lato della pipetta 5 mL per evitare il curling durante la centrifugazione. Le cellule sono permesso di raggiungere 1.000 giri al minuto e si fermò. Trapianto: topi destinatari sono anestetizzati, rasato e pulito. Una piccola incisione viene fatta sul fianco sinistro del mouse e il rene è esposto. L&#39;, rene grasso e il tessuto sono mantenute umide con normale soluzione salina tampone. L&#39;estremità distale del PE50 è attaccato ad una siringa Hamilton azionamento della vite, contenente un puntale, utilizzando il tubo adattatore silicone. Un piccolo nick è fatto sul lato fianco destro del rene, non troppo grande né troppo profondo. L&#39;estremità smussata del tubo PE50, più vicino alle celle, è accuratamente posizionato sotto la capsula, il tubo viene spostato delicatamente per fare spazio, mentre tampone soluzione fisiologica; una capsula secca può strappare facilmente. Una piccola bolla d&#39;aria è fornito sotto la capsula lentamente componendo il disco vite siringa. Isolotti sono poi lentamente consegnati dietro la bolla d&#39;aria. Omeostasi renale una volta le isole sono state consegnate viene mantenuta e la knick è cauterizzato a basse temperature. Il rene è posto nuovamente dentro la cavità ed il peritoneo e la pelle sono suturate e pinzati. I topi sono immediatamente trattati con Buprenorfina e Flunixin mq e posto in una gabbia su una piastra elettrica.

Watch this Scientific Journal Video about Transplantation of Pancreatic Islets Into the Kidney Capsule of Diabetic Mice at JoVE.com

Transplantation of Pancreatic Islets Into the Kidney Capsule of Diabetic Mice

Il nostro protocollo è stato sviluppato per fornire in modo pulito e facilmente le cellule o isolotti sotto la capsula renale di topi. Le cellule sono concentrate in pellet nel tubo finale utilizzato per il trapianto delle cellule sotto la capsula renale. La facilità di questa tecnica riduce lo stress per le cellule e il mouse.

Il trapianto di isole pancreatiche nella capsula renale di topi diabetici

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice.  We found that it was easier ...

transplantation-pancreatic-islets-into-kidney-capsule-diabetic

Research

JoVE Journal

Biology

27.2K Views.  University of California, San Francisco - UCSF. Il nostro protocollo è stato sviluppato per fornire in modo pulito e facilmente le cellule o isolotti sotto la capsula renale di topi. Le cellule sono concentrate in pellet nel tubo finale utilizzato per il trapianto delle cellule sotto la capsula renale. La facilità di questa tecnica riduce lo stress per le cellule e il mouse.

Video: Transplantation of Pancreatic Islets Into the Kidney Capsule of Diabetic Mice

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.
Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

Un metodo per isolamento Islet murini e trapianto renale subcapsulare

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

Ricerca

Medicina

Scaffold-supported Transplantation of Islets in the Epididymal Fat Pad of Diabetic Mice

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy may incur acute whole blood reactions and result in poor islet engraftment. Here, we report a robust protocol for the transplantation of islets at the extrahepatic transplantation site-the epididymal fat pad (EFP)-in a diabetic mouse model. A protocol to isolate and purify islets at high yields from C57BL/6J mice is described, as well as a transplantation method performed by seeding islets onto a decellularized scaffold (DCS) and implanting them at the EFP site in syngeneic C57BL/6J mice rendered diabetic by streptozotocin. The DCS graft containing 500 islets reversed the hyperglycemic condition within 10 days, while the free islets without DCS required at least 30 days. The normoglycemia was maintained for up to 3 months until the graft was explanted. In conclusion, DCS enhanced the engraftment of islets into the extrahepatic site of the EFP, which could easily be retrieved and might provide a reproducible and useful platform for investigating the scaffold materials, as well as other transplantation parameters required for a successful islet engraftment.

This protocol demonstrates murine islet isolation and seeding onto a decellularized scaffold. Scaffold-supported islets were transplanted into the epididymal fat pad of streptozotocin (STZ)-induced diabetic mice. Islets survived at the transplantation site and reversed the hyperglycemic condition.

Il trapianto di isole nel supporto del grasso epididimico dei topi diabetici

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy ...

Bioingegneria

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the availability of the widest range of molecular probes and reagents to perform in vivo as well as in vitro studies. Based on our experience with various murine transplantation models, we developed a heterotopic pancreas transplantation model in mice with the intent to analyze mechanisms underlying severe ischemia reperfusion injury-associated early graft damage. In contrast to previously described techniques using suture techniques, herein we describe a new procedure using a non-suture cuff technique. 
In recent years, we have performed more than 300 pancreas transplantations in mice with an overall success rate of &gt;90%, a success rate never described before in mouse pancreas transplantation. The backbone of this non-suture cuff technique for graft revascularization consists of two major steps: (I) pulling the recipient vessel over a polyethylene/polyamide cuff and fixing it with a circumferential ligature, and (II) placing the donor vessel over the everted recipient vessel and fixing it with a second circumferential ligature. The resultant continuity of the endothelial layer results in less thrombogenic lesions with high patency rates and, finally, high success rates.
In this model, arterial anastomosis is achieved by pulling the abdominal aorta of the donor graft over the everted common carotid artery of the recipient animal. Venous drainage of the graft is achieved by pulling the portal vein of the graft over the everted external jugular vein of the recipient. This manuscript provides details and crucial steps of the organ recovery and organ implantation procedures, which will allow researchers with microsurgical skills to perform the transplantation successfully in their laboratories.

Among abdominal solid organ transplantation, pancreatic grafts are prone to develop severe ischemia reperfusion injury-associated graft damage, leading eventually to early graft loss. This protocol describes a model of murine pancreas transplantation using a non-suture cuff technique, ideally suited for analyzing these early, deleterious damages.

Modello di topo per il trapianto di Pancreas usando una tecnica modificata polsino

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the ...

Intraportal Transplantation of Pancreatic Islets in Mouse Model

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation site in experimental islet transplantation is the kidney capsule. However, as less is known about the interaction of pancreatic islets with blood constituents, it also makes sense to utilize the portal vein approach in experimental islet transplantation.
This protocol demonstrates an intraportal islet transplantation technique in NMRI nude mice. Streptozotocin (180 mg/kg) is injected intraperitoneally to induce hyperglycemia in recipient mice. They are considered as diabetic at a non-fasting blood glucose level greater than 20 mmol/L. One day prior to transplantation, mouse pancreatic islets are isolated from the donor pancreas by collagenase digestion; a minimum of 350 islets are utilized per diabetic recipient. Depending upon the islet isolation yield, two or more donor mice are utilized per recipient. After overnight culture at 37 &#176;C, islets are administered into the recipient liver via the portal vein. After surgery, the mice are protected in red Makrolon houses and observed until are awake. This protocol maintains glycemic control for 120 days in syngeneic mice and 15 days in allogeneic mice.

Pancreatic islet transplantation is a way to achieve normoglycemia in type 1 diabetes. This article focuses on the transplantation technique through the intraportal route to maintain normal glucose levels in diabetic mice.

Intraportal trapianto di isole pancreatiche nel modello del topo

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation ...

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The &#946;-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the &#946;-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on &#946;-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased &#946;-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

Assaying in vitro &#946;-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many&#160;downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining &#946;-cell function.

Un metodo per l&#39;isolamento del mouse isole pancreatiche e intracellulare cAMP Determinazione

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

Biologia

Inguinal Subcutaneous White Adipose Tissue (ISWAT) Transplantation Model of Murine Islets

Pancreatic islet transplantation is a well-established therapeutic treatment for type 1 diabetes. The kidney capsule is the most commonly used site for islet transplantation in rodent models. However, the tight kidney capsule limits the transplantation of sufficient islets in large animals and humans. The inguinal subcutaneous white adipose tissue (ISWAT), a new subcutaneous space, was found to be a potentially valuable site for islet transplantation. This site has better blood supply than other subcutaneous spaces. Moreover, the ISWAT accommodates a larger islet mass than the kidney capsule, and transplantation into it is simple. This manuscript describes the procedure of mouse islet isolation and transplantation in the ISWAT site of syngeneic diabetic mouse recipients. Using this protocol, murine pancreatic islets were isolated by standard collagenase digestion and a basement membrane matrix hydrogel was used for fixing the purified islets in the ISWAT site. The blood glucose levels of the recipient mice were monitored for more than 100 days. Islet grafts were retrieved at day 100 after transplantation for histological analysis. The protocol for islet transplantation in the ISWAT site described in this manuscript is simple and effective.

Inguinal Subcutaneous White Adipose Tissue ISWAT Transplantation Model of Murine Islets

In this protocol, a method of murine islet isolation and transplantation into the inguinal subcutaneous white adipose tissue is described. Isolated syngeneic murine islets are transplanted into a murine recipient using a basement membrane hydrogel. The blood glucose level of the recipients is monitored, and histology analysis of the islet grafts is performed.

Modello di trapianto di Murine (ISWAT) del tessuto adiposo sottocutaneo inguinale (ISWAT)

Pancreatic islet transplantation is a well-established therapeutic treatment for type 1 diabetes. The kidney capsule is the most commonly used site for ...

Longitudinal In Vivo Imaging and Quantification of Human Pancreatic Islet Grafting and Contributing Host Cells in the Anterior Eye Chamber

Imaging beta cells is a key step towards understanding islet transplantation. Although different imaging platforms for the recording of beta cell biology have been developed and utilized in vivo, they are limited in terms of allowing single cell resolution and continuous longitudinal recordings. Because of the transparency of the cornea, the anterior chamber of the eye (ACE) in mice is well suited to study human and mouse pancreatic islet cell biology. Here is a description of how this approach can be used to perform continuous longitudinal recordings of grafting and revascularization of individual human islet grafts. Human islet grafts are inserted into the ACE, using NOD.(Cg)-Gt(ROSA)26Sortm4-Rag2-/-mice as recipients. This allows for the investigation of the expansion of recipient versus donor cells and the contribution of recipient cells in promoting the encapsulation and vascularization of the graft. Further, a step-by-step approach for image analysis and quantification of the islet volume or segmented vasculature and islet capsule forming recipient cells is outlined.

The goal of this protocol is to continuously monitor the dynamics of the human pancreatic islet engraftment process and the contributing host versus donor cells. This is accomplished by transplanting human islets into the anterior chamber of the eye (ACE) of an NOD.(Cg)-Gt(ROSA)26Sortm4-Rag2-/-mouse recipient followed by repeated 2-photon imaging.

Longitudinale in Vivo Imaging e quantificazione dell'isolotto pancreatico umano Innesto e contribuendo cellule ospitanti nella camera occhio anteriore

Imaging beta cells is a key step towards understanding islet transplantation. Although different imaging platforms for the recording of beta cell biology ...

Minimizing Post-Infusion Portal Vein Bleeding during Intrahepatic Islet Transplantation in Mice

Although the liver is currently accepted as the primary transplantation site for human islets in clinical settings, islets are transplanted under the kidney capsule in most rodent preclinical islet transplantation studies. This model is commonly used because murine intrahepatic islet transplantation is technically challenging, and a high percentage of mice could die from surgical complications, especially bleeding from the injection site post-transplantation. In this study, two procedures that can minimize the incidence of post-infusion portal vein bleeding are demonstrated. The first method applies an absorbable hemostatic gelatin sponge to the injection site, and the second method involves penetrating the islet injection needle through the fat tissue first and then into the portal vein by using the fat tissue as a physical barrier to stop bleeding. Both methods could effectively prevent bleeding-induced mouse death. The whole liver section showing islet distribution and evidence of islet thrombosis post-transplantation, a typical feature for intrahepatic islet transplantation, were presented. These improved protocols refine the intrahepatic islet transplantation procedures and may help laboratories set up the procedure to study islet survival and function in pre-clinical settings.

Here we present refined surgical procedures on successfully performing intraportal islet transplantation, a clinically relevant but technically challenging surgical procedure, in mice.

Ridurre al minimo il sanguinamento della vena porta post-infusione durante il trapianto di isole intraepatiche nei topi

Although the liver is currently accepted as the primary transplantation site for human islets in clinical settings, islets are transplanted under the ...

A Method for Islet Transplantation to the Omentum in Mouse

Islet transplantation has been proposed to be a potential treatment for type 1 diabetes. Recent compelling evidence indicates that intravascular islet infusion is far from ideal and therefore, the omentum is re-emerging as a potentially valuable site for islet transplantation. This experiment requires the isolation of high quality islets and the implantation of the islets to the diabetic recipients. Transplantation to the omentum requires surgical steps that can be better demonstrated visually. Here, the detailed steps for this procedure are presented. Two methods of mixing the isolated islets with hydrogel before placing the mixture into the omental pouch of diabetic mice are described here. Different hydrogels are used for the different conditions. Blood glucose levels of diabetic mouse recipients of syngeneic islets in the omentum were monitored for up to 35 days. Some animals were sacrificed after 14 days to perform immuno-histochemical analysis. This pre-clinical transplantation approach can be used as preliminary data leading up to translation to clinical transplantation.

A method for the omental transplantation of islets in a mouse is introduced. The isolated islets are mixed with hydrogel and the mixture is placed into the omental pouch of a diabetic mouse. Then, the blood glucose is monitored, and immuno-histochemical analysis is performed.

Un metodo per il trapianto dell'isolotto al omento in Mouse

Islet transplantation has been proposed to be a potential treatment for type 1 diabetes. Recent compelling evidence indicates that intravascular islet ...

Grafting Pancreatic Islets: A Method to Implant Pancreatic Islets into the Anterior Chamber of the Mouse Eye for In Vivo Imaging

Source: Nilsson, J.et. al.&#160;<a href="https://www.jove.com/v/61234/" target="_blank">A Longitudinal In Vivo Imaging and Quantification of Human Pancreatic Islet Grafting and Contributing Host Cells in the Anterior Eye Chamber</a>. J. Vis. Exp. (2020).
This video describes the protocol for grafting human pancreatic islets into the anterior chamber of the mouse&#8217;s eye to monitor revascularization of human islet grafts in vivo. It also helps to understand the contribution of recipient versus donor cells in promoting the encapsulation and vascularization of the graft.

Innesto di isole pancreatiche: un metodo per impiantare le isole pancreatiche nella camera anteriore dell'occhio del topo per l'imaging in vivo

Source: Nilsson, J.et. al.&#160;A Longitudinal In Vivo Imaging and Quantification of Human Pancreatic Islet Grafting and Contributing Host Cells in the ...